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Thirkell, T., Cameron, D. orcid.org/0000-0002-5439-6544 and Hodge, A. (2019)

Contrasting nitrogen fertilisation rates alter mycorrhizal contribution to barley nutrition in a

field trial. Frontiers in Plant Science, 10. 1312.

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doi: 10.3389/fpls.2019.01312

Contrasting Nitrogen Fertilisation

Rates Alter Mycorrhizal Contribution

to Barley Nutrition in a Field Trial

Tom Thirkell 1,2*, Duncan Cameron 2 and Angela Hodge 1

1 Department of Biology, University of York, York, United Kingdom, 2 Department of Animal and Plant Sciences, University of

Shefield, Shefield, United Kingdom

Controlled environment studies show that arbuscular mycorrhizal fungi (AMF) may contribute to plant nitrogen (N) uptake, but the role of these near-ubiquitous symbionts in crop plant N nutrition under natural field conditions remains largely unknown. In a field trial, we tested the effects of N fertilisation and barley (Hordeum vulgare L.) cultivar identity on the contribution of AMF to barley N uptake using 15N tracers added to rhizosphere soil

compartments. AMF were shown capable of significantly increasing plant 15N acquisition

from root exclusion zones, and this was influenced by nitrogen addition type, N fertiliser application rate and barley cultivar identity. Our data demonstrate a previously overlooked potential route of crop plant N uptake which may be influenced substantially and rapidly in response to shifting agricultural management practices.

Keywords: arbuscular mycorrhiza, nitrogen, barley, field trial, plant ecophysiology

INTRODUCTION

Nitrogen (N) is usually the most limiting mineral nutrient to plant growth (Agren et al., 2012) and maintaining modern agricultural production requires frequent and substantial application of fertiliser to farm soils. In various forms an estimated 50 MT year−1 fertiliser N is applied to

agricultural land worldwide (Ladha et al., 2016). Assimilation of applied N by crops may be under 50% (Ladha et al., 2005, Masclaux-Daubresse et al., 2010); a signiicant fraction of this applied N is wasted — lost through processes including volatilisation, microbial immobilisation, runof, and leaching (Ladha et al., 2016, Cameron et al., 2013). here is economic and ecological pressure on farmers to optimise the N uptake eiciency of crop plants (Hawkesford, 2014) and by reducing the reliance on non-renewable inputs, improve the sustainability of agriculture (Pretty, 2008). his progress will require the integration of biological and ecological processes into agriculture, and better understanding of soil microbial communities and their roles in nutrient cycling (Rillig et al., 2016, Pretty, 2018).

As near-ubiquitous symbionts of cereal crops, arbuscular mycorrhizal fungi (AMF) are prime targets to investigate the role of soil biota in improving agricultural sustainability (Gosling et al., 2006, hirkell et al., 2017, Rillig et al., 2019). he majority of land plant species engage in symbiosis with these fungi, which may aid plants’ mineral nutrient uptake from soils, in exchange for photosynthetic carbon (C) from their plant hosts (Smith and Read, 2008). he inluence that AMF mycelia may exert over nutrient dynamics in agricultural systems is not limited to direct efects on plant nutrient acquisition however; the presence of AMF has been shown to reduce mineral fertiliser leaching (Cavagnaro et al., 2015) and to inluence greenhouse gas emissions (Storer et al., 2018). While the role of AMF in biogeochemical cycles is undoubtedly complex, of pressing need is

Edited by: Paola Bonfante, University of Turin, Italy

Reviewed by: Jan Jansa, Institute of Microbiology (ASCR), Czechia Erik Verbruggen, University of Antwerp, Belgium *Correspondence: Tom Thirkell t.j.thirkell@leeds.ac.uk †Present address: Tom Thirkell, Centre for Plant Sciences, University of Leeds, United Kingdom

Specialty section: This article was submitted to Plant Microbe Interactions, a section of the journal Frontiers in Plant Science

Received: 20 May 2019 Accepted: 20 September 2019 Published: 30 October 2019 Citation: Thirkell T, Cameron D and Hodge A (2019) Contrasting Nitrogen Fertilisation Rates Alter Mycorrhizal Contribution to Barley Nutrition in a Field Trial. Front. Plant Sci. 10:1312. doi: 10.3389/fpls.2019.01312

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Mycorrhiza-Mediated Barley Nitrogen Nutrition Thirkell et al.

to determine the extent to which plants rely on these symbionts for mineral nutrient acquisition.

It is well established that AMF can contribute to plant N uptake (Ames et al., 1983; Hodge et al., 2001; Leigh et al., 2009; hirkell et al., 2016), but the extent to which this takes place, and whether it is ecologically or agriculturally relevant is unclear (Smith and Smith, 2011a). his is in part due to relatively little experimental attention. here remains in the literature a focus on the role of AMF in plant phosphorus (P) uptake (Smith and Smith, 2011a;

Karasawa et al., 2012; Ezawa and Saito, 2018), and consideration of symbiotic N uptake is oten restricted to diazotrophic bacteria while AMF are oten overlooked (Garcia et al., 2016).

Improved access to poorly-mobile soil P is, in most instances, the primary beneit of AMF to their plant hosts (Smith and Read, 2008). he relative immobility of inorganic P (Pi) in soil means that plant uptake of Pi from the rhizosphere can outpace Pi difusion from the surrounding bulk soil and the subsequent P-depletion zones that form around the root are narrow and sharply deined. By engaging in symbiosis with AMF, with a mycelium spreading several centimetres beyond the rhizosphere, the plant efectively increases the volume of soil from which it can acquire nutrients, particularly poorly mobile ions such as Pi (Sanders and Tinker,

1973; Hodge, 2017). Nitrate (NO3−) and ammonium (NH4+),

the predominant forms in which plants and fungi acquire N

(Marschner, 2011), are more mobile in soil than orthophosphate

(Tinker and Nye, 2000). Despite this, a zone of N-depletion may

still form around the root (Brackin et  al., 2017), in which case AMF may facilitate improved N capture for their plant hosts. With smaller diameters than plant roots, AMF hyphae may also penetrate soil micropores more efectively than a plant root, and thereby be present when inorganic N forms are released through microbial decomposition processes and efectively scavenge for this released inorganic N (Hodge, 2014).

Results from microcosm studies are conlicting as to the importance of AMF in plant N uptake (Hodge and Storer, 2015). While a number of studies have shown no improvement of N uptake by AM plants versus non-mycorrhizal counterparts

(Cui and Caldwell, 1996a; Cui and Caldwell, 1996b; Reynolds

et al., 2005;Kahkola et al., 2012), it is possible that AMF make an invisible contribution to nutrient acquisition which cannot easily be identiied without the use of isotope tracing techniques. Mycorrhizal downregulation of plant root phosphate transporters has been identiied in a number of studies (Smith et al., 2003;

Smith et al., 2004). In this situation, AMF may be responsible

for the majority of a plant’s P acquisition, but root transporter downregulation may result in reduced plant P uptake compared to non-mycorrhizal control plants (Smith et al., 2003; Smith et al., 2004). Whether a similar phenomenon occurs in mycorrhizal root N uptake remains unclear. Isotope tracing data does, however, show that AMF can transfer substantial amount of N to a host plant (Leigh et al., 2009; hirkell et al., 2016), while the contribution of AMF to ield-grown plant N uptake is unknown.

AMF are capable of acquiring N from decomposing organic sources (Leigh et al., 2009; Hodge and Fitter, 2010; Herman et al 2012; Barrett et al., 2014; hirkell et al., 2016) and even to acquire some organic N directly from the hyphosphere, notably as amino acids (Hawkins et al., 2000; Breuninger et al., 2004; Whiteside et al.,

2012a; Whiteside et al., 2012b, Tisserant et al., 2012) and perhaps as dipeptides (Belmondo et al., 2014). As in plants however, the vast majority of N acquired by AMF is thought to be as NO3− or NH4+

(Govindarajulu et al., 2005; Bucking and Kale, 2015). Greater N uptake as NO3− might be expected as it is usually more abundant

than NH4+ because of rapid nitriication (Marschner, 2011).

However, because N acquired as NO3− must be reduced to NH4+

before further assimilation, it should be energetically favourable for AMF to acquire N as NH4+ (Hodge et al., 2010; Courty et al., 2015).

Corroborative data remains equivocal as to AMF “preference” for N types (Johansen et al., 1993; Hawkins and George, 2001). As NO3− and NH4+ are the most commonly-used forms of fertiliser

in Western agriculture, the need to understand mycorrhizal plant acquisition of these N sources is pressing.

Nutrient trade between partners in AM symbioses shows considerable variation in response to biotic factors such as plant and fungal genotype (Smith et al., 2004), in addition to abiotic factors including soil nutrient status (Johnson, 2010; Johnson et al., 2015). Despite substantial experimental data, predictability of the extent to which plants beneit from AMF colonisation remains poor. For example, no universally beneicial fungal isolate has been identiied and comparatively few plants are obligate symbionts with AMF.

Despite the widespread distribution of AMF (Smith and Read,

2008; Davison et al., 2015) and the readiness with which they

colonise most staple crop plant roots (Smith and Smith, 2011a), little is understood about the function of AMF in the ield (Lekberg and Helgason, 2018; Ryan and Graham, 2018). Most published material on the function of AMF is derived from studies conducted under controlled conditions, oten comparing AM plants with non-AM controls. While such experiments have provided much valuable data and insight, their indings cannot directly be extrapolated to the ield scale, as the occurrence of non-AM cereals in most arable soils is unlikely (Smith and Smith, 2011a). Despite disruptive practices such as tilling and the application of fungicides, there remains a substantial AMF spore bank (and therefore inoculum potential) in agricultural soils (Sosa-Hernandez et al., 2018) and it is very likely that plants in arable ield soil will be colonised by AMF (Smith and

Smith, 2011a). Further research is needed to begin to understand

how AMF might afect crop plant nutrient uptake in situ.

Adding 15N isotope tracers to mesh-walled soil compartments

in a ield trial, we examined the role of AMF in the N acquisition by barley (Hordeum vulgare L.) cultivars “Meridian” and “Maris Otter”. Isotopic 15N labelling was carried out in plots receiving contrasting

N rates to test the impact of N availability on nutrient transfer in the symbiosis. We tested the hypothesis that increased N fertilisation would result in more AMF transfer of N to host plants because AMF, by virtue of their size, would be better able than roots to compete with the soil microbiome for the added N held in physically small microsites. N tracers were added as NH4+ or NO3− to investigate the

relative uptake and transfer of diferent N sources by AMF.

MATERIALS AND METHODS

Field Trial Design

Data were gathered from a larger ield trial, designed and implemented at Sancton, East Riding of Yorkshire (co-ordinates

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53°51′10.2″N 0°35′29.1″W), by ADAS (Pendeford, Wolverhampton, UK). he ADAS trial was set up to test how barley yield compares among 6 application rates of ammonium nitrate (NH4NO3) fertiliser (Nitram, CF Fertiliser, Ince, Cheshire,

UK) ranging from 0–300 kg ha−1. he soil at the trial site

comprises a silty rendzina, with a signiicant proportion of chalk fragments (UKSO, 2016). Soil mineral N, quantiied shortly before sowing, was 29.9 kg N ha−1, of which 28 kg was nitrate-N

and 1.9 kg ammonium-N. he ield site on which the trial was based is a commercial arable farm, with barley (Hordeum vulgare L.), oilseed rape (Brassica napus L.) and wheat (Triticum aestivum L.) grown in a rotation.

he ADAS trial used plots measuring 12 m × 1.5 m, clustered in groups of 6 by N application rate, with each variety represented once per cluster. Each N application rate was applied to 3 replicate clusters, of 6 varieties, meaning 18 clusters in total, with a combined area of 1944 m2. Experimental clusters of N

application rates were separated to each side by bufer zones 6 m wide, and at each end by bufer zones 3 m long (Figure 1). Owing to the logistical challenges of sampling the entire trial, the experimental work presented here is gathered from two of the N application rates (60 kg ha−1 (N rate 2 in Figure 1), and 280 kg

ha−1 (N rate 5 in Figure 1)), and two of the barley cultivars: KWS

Meridian (KWS UK Ltd, hriplow, Hertfordshire, UK), a 6-row feedstock barley; and Maris Otter (Robin Appel, Waltham Chase, Hampshire, UK), a 2-row malting barley, giving 4 treatment groups, with 3 replicate plots per treatment. Meridian and Maris Otter were chosen from the panel of 6 cultivars available in the trial as they represent contrasting ages of barley varieties, developed in the 1960s and 2000s respectively. Further, Maris Otter is a malting barley, characterised by a low grain protein content, while Meridian was developed as a feedstock barley, with

a higher grain protein (and therefore N) content. Experimental sampling and isotope labelling were carried out during the post-anthesis, grain illing period — approximate growth stages 70–80 (Zadoks et al., 1974).

Intraradical and Extraradical AMF

Quantification

AMF colonisation of both barley varieties was conirmed and then quantiied by staining of roots collected from the trial plots. Roots were collected from between 5 and 15 cm below the surface. Ater clearing in 10% (w/v) KOH for 20 min at 70°C, roots were rinsed in de-ionised water, acidiied in 1% (v/v) HCl at 25°C for 10 min and then stained in Trypan Blue at 25°C for 20 min. Roots were then rinsed again in de-ionised water before being let in a 50% (v/v) glycerol solution for 24 h, before being mounted onto microscope slides to allow quantiication of root length colonisation (RLC) using the gridline intersect method (McGonigle et al., 1990).

Soil samples were collected from between 5 and 15 cm below the soil surface. As AMF hyphal turnover can be rapid (Staddon et al., 2003), hyphal extraction took place within 6 h of collection to minimise loss due to decomposition. Extraradical hyphal quantity in the plots was determined using an adapted method from Staddon et al. (1999). Briely, samples of known mass (5–10 g) were suspended in 500 mL of de-ionised water and agitated with a magnetic stirrer plate in order to free the hyphae from soil particles. From this, 200 mL was decanted to a smaller beaker on a magnetic stirrer. Aliquots (10 mL) were removed and vacuum iltered through 0.45 µm nylon mesh (Anachem, Bedfordshire, UK) and hyphal length density (HLD) was quantiied using the gridline intersect method (Hodge, 2001).

FIGURE 1 | ADAS experiment established at Sancton, East Riding of Yorkshire, UK. Six barley (Hordeum vulgare L.) cultivars were planted at the trial site, and received one of 6 N addition rates, ranging from 0 to 300 kg ha−1. Each combination of barley cultivar and N rate was replicated 3 times. Each plot has 3 numbers,

denoting: plot identity, N addition rate and barley cultivar, reading top to bottom. Nitrogen addition rate “2” represents 60 kg ha−1 and “5” is 280 kg ha−1. Plot colours

also represent N addition rate. Meridian barley is denoted by “4” and Maris Otter by “5”. Asterisks (*) represent plots from which root samples were taken for analysis of root length colonisation and to which 15N tracer was added. Reproduced with permission from Pete Berry and Kate Storer, ADAS.

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Mycorrhiza-Mediated Barley Nitrogen Nutrition Thirkell et al.

15

N Stable Isotope Labelling

he AMF contribution to barley N uptake was investigated by adding a solution of 15N (as either (15NH

4)2SO4 or K15NO3), into

mesh-walled cores, into which AMF hyphae could access but plant roots could not, or (as controls for difusion and mass low of the added N) cores into which neither AMF hyphae or roots could access. Isotopic 15N was added in the form of Long Ashton nutrient

solution (LAS) (Smith et al., 1983), which can be prepared variously to provide 15N as 15NH

4+ or 15NO3− in equimolar concentrations.

he LAS was made to the standard protocols except N being 300% the original concentrations. Each core received 5 mL of LAS, containing 0.683 mg 15N. (Long Ashton nutrient solution protocol

is included in Supplementary Information Document 1). Hyphal access cores were constructed following an adapted method from Johnson et al. (2001). Lengths of PVC tubing (length 85 mm, internal diameter 13 mm, external diameter 16 mm; internal volume 9.9 cm3) with 2 windows cut in the sides of

the lower 2/

3 of the tube so that 50% of the side area was open, were

wrapped in a 20 µm nylon mesh (John Stanier and Co., Whiteield, Manchester, UK), ixed with Tensol adhesive cement (Bostik Inc., Wauwatosa, Wisconsin, USA). he open bottom end of each tube was covered with the same size mesh. Control cores, which allowed difusion and mass low of solutes but prevent hyphal ingrowth, were covered with 0.45 µm nitrocellulose membrane mesh to prevent root and hyphal ingrowth. Cores were illed with a 1/1 (v/v) mixture of silica sand and TerraGreen®

(calcinated attapulgite clay, Oil-Dri, Cambridgeshire, UK), which had been sterilised by autoclaving (121°C for 44 min), providing a uniform substrate into which the 15N solutions could be added.

Each of these cores was then placed inside another, slightly larger core, constructed in the same manner (length 75 mm internal diameter 18 mm, external diameter 21 mm). hese cores were also covered in a 20 µm nylon mesh. Such a “core in a core” design allows the placement of zones of deined and uniform size

into the soil, to which 15N label solutions could be added. A small

(approximately 1 mm) air gap is made between the external mesh wall of one core and the internal mesh wall of the other, which should reduce the rapid difusion of N from the site of addition, which has been a problem in studies where 15N has been added

(Smith and Smith, 2011b). Difusion and mass low are unlikely

to be prevented entirely, as the pressure of soil on the sides of the core may push the mesh together so that the two layers of mesh make contact. However, the system provides a more stable labelling zone than using a single core, where one mesh layer may be easily damaged (Johnson et al., 2001).

Each of the 12 experimental plots received four cores (1. No AMF Access + 15NH

4+; 2. AMF Access + 15NH4+; 3. No AMF

Access + 15NO

3−; 4. AMF Access + 15NO3−), spaced 3 m apart to

avoid contamination of 15N from neighbouring cores (Figure 2).

Placement of cores took place 8 weeks before label addition to allow hyphal ingrowth from the bulk soil. A piece of tape was placed over the top of cores to minimise contamination. his tape was removed for 15N addition and then replaced.

Sample Collection and Preparation

Ater 7 days, the nearest plant to each labelling core was cut at ground level and removed, dried at 70°C for 48 h and homogenised in a kitchen blender (Morphy Richards, Mexborough, South Yorkshire, UK) then in a ball mill (MM400 Ball Mill, Retsch GmbH, Haan, Germany). Homogenised shoot samples of known mass (3 mg ± 0.5 mg) were used to quantify 15N and N content,

performed by isotope ratio mass spectrometry (IRMS) (PDZ 2020, Sercon Ltd, Crewe, UK).

Statistical Analysis

For all data, statistical analysis was performed using the “R 3.1.0” statistical package, through the “RStudio” integrated development

FIGURE 2 | Diagram of 15N addition experiment. PVC cores were inserted adjacent to barley (Hordeum vulgare L.) plants, four cores per plot, spaced 3 m apart.

Cores were organised as follows A1 – AMF Access + Ammonium (NH4+); A2 - No AMF Access + Nitrate (NO3−); A3 - AMF Access + NO3−; A4 - No AMF Access +

NH4+ Each core received 0.683 mg 15N added as Long Ashtons nutrient solution. Plant shoots closest to the core (B1-4) were removed, dried and homogenised for

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environment (R foundation for Statistical Computing, Vienna, Austria). Data were tested for normality using Shapiro-Wilk and Kolmogorov-Smirnov tests, and Levene’s test was used to conirm homogeneity of variance. Where these tests suggested data did not match test assumptions, data were square-root or log-transformed prior to analysis. Data for root length colonisation, hyphal length density, barley N concentration and biomass were tested by two-way ANOVA, using N addition rate and barley variety as explanatory variables. As two additional explanatory variables were added in the trial for 15N uptake (N addition type,

ammonium/nitrate; AM treatment, access/no access), and the small number of replicates in the ADAS ield trial, it was not possible to test these factors and the N addition rate and barley cultivar at once. As such, data were split into barley cultivar and N application rate for the 15N data and tested by two-way ANOVA.

Here, 15N enrichment was the response variable, while N type

and AMF access treatment were the explanatory variables.

RESULTS

Shoot acquisition of 15N added to mesh cores was signiicantly

improved by allowing AMF access into cores, but only when added as 15NO

3−, and only in the High-N plots of Meridian barley (Figure

3). T-tests indicate that only in High-N Meridian plots receiving

15NO

3− were 15N enrichment levels greater in the AM access

treatment than in the no access controls (T2 = 4.48, p = 0.023)

(Supplementary Information, Figure 1). A two-way ANOVA showed that in High-N Meridian, there was a signiicant efect of N source (F1,8 = 12.73, p = 0.007) and AMF access to cores (F1,8 =

27.86, p = 0.007). here was also a signiicant interaction between N source and AMF access (F1,8 = 14.25, p = 0.005) (Figure 3). In

High-N Meridian with AMF access, the harvested plants, i.e. those individuals closest to the core to which the isotope label was added, acquired on average 1.62% of the 15N supplied. Other treatment

groups saw no greater plant uptake of 15N where AMF could access

the isotope label than in no-access controls. Excepting High-N Meridian plots, mean shoot 15N content did not difer among

treatments and controls, indicating similar plant acquisition of N following difusion/mass low out and into the soil, but minimal fungal-mediated uptake.

All plant roots studied were found to be colonised by AMF, indicating a substantial inoculum potential of the soil at the trial site, although no diferences were found between cultivar or N-rate treatments (p > 0.05). Mean colonisation was 33.7% (± 3.52% SEM) across all treatment groups (Figure 4). Extraradical mycelium (ERM) hyphal densities, measured in the zones to which 15N was added, were not diferent among

treatment groups (p > 0.05). Mean ERM hyphal density across all treatments was 2.49 m g−1 DW soil (± 0.31 m g−1 SEM). In

both cultivars, High-N plots supported ~ 60% higher shoot N content than Low-N plots (F1,8 = 74.55, p < 0.001), and shoot N

concentration was signiicantly higher in High-N than Low-N plots (F1,8 = 84.28, p < 0.001). Mean shoot N concentration was

9.30 mg g−1 DW in Low-N blocks of Maris Otter, and 14.75 mg

g−1 DW in the High-N. Meridian showed a very similar trend,

as N concentration increased from 9.57 mg g−1 DW in Low-N

plots to 14.38 mg g−1 DW in the High N. Shoot N concentration

and content did not difer between the two cultivars tested. Shoot DW did not difer between the varieties or the N addition rates.

FIGURE 3 | Excess 15N content in Maris Otter and Meridian shoots (calculated by subtracting shoot 15N content in each “Access” unit from the mean of the

corresponding values in the “No Access” units). Shoot 15N enrichment was significantly higher than “no access” controls when supplied as nitrate to Meridian barley

in High-N plots. Circles represent individual data points, boxplot centre bars represent the median values. High-N + ammonium groups are represented by white bars, High-N + nitrate by light blue bars, Low-N + ammonium by dark grey bars and Low-N + nitrate by dark blue bars. Data shown are means ± SEM, n = 3. Bars sharing the same letter are not significantly different.

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Mycorrhiza-Mediated Barley Nitrogen Nutrition Thirkell et al.

DISCUSSION

he enrichment of 15N in barley shoots suggests a role for

AMF-facilitated N acquisition by crop plants, a phenomenon not previously observed in a ield setting. Moreover, our data suggest this route of N uptake is dependent upon barley cultivar identity, the N form added and the rate at which N has previously been applied to the plots. AMF have been shown to transfer substantial quantities of N to plants in root organ culture experiments (Jin et al., 2005) although caution must be exercised before extrapolating these values to crop plant systems as they are far-removed from realistic mycorrhizal physiology. Whole-plant microcosm studies conducted under greenhouse conditions have given mixed results as to whether AMF may contribute to plant N nutrition (Hodge and Storer, 2015). Our data provide the irst suggestion that AMF may have a role in cereal crop N uptake in the ield. Our data also suggest that short-term changes in N fertilisation regimes can elicit shits in AM functioning.

While our data suggest a preference for AMF to transfer N to plants when provided to this system as NO3− rather

than NH4+, previous experimental evidence as to inorganic N

source preference by AMF is equivocal (Johansen et al., 1993;

Hawkins and George, 2001). Higher uptake of NO3− than NH4+

is contrary to models which suggest NH4+ acquisition should be

less energetically expensive (Govindarajulu et al., 2005). Hyphal NH4+ uptake may be retarded by problems of charge balancing

that are perhaps not encountered when N is acquired as NO3−.

Simultaneous uptake of NO3− and cations such as K+, Ca2+ or Mg2+

from the soil may avoid changes in electrochemical potential across exchange surfaces, allowing N acquisition. Meanwhile, NH4+ uptake would require proton secretion (or anion uptake),

which may shit soil pH making further NH4+ uptake more

diicult. Nitrate-N comprised over 90% of the available N in the soil before the trial was planted, a trend which is not unusual, as NO3− oten dominates inorganic N pools in arable soils

(Marschner, 2011). hese relative abundances of N sources may

have led to AMF hyphal physiology being acclimated to nitrate uptake (Garraway and Evans, 1984), meaning suddenly-available NH4+ could not be acquired efectively. Although the movement

and cycling of nitrate and ammonium are known to be inluenced by soil moisture (Homyak et al., 2017), precipitation data for the site (Supplementary Information, Table 1) indicates no extraordinary rainfall in the weeks over which the experiment took place, suggesting this was of minor importance here.

While recovery of only 1.6% of the 15N label seems low, total 15N

recovery is likely to have been greater than the data suggests. Our data are derived from the aboveground tissue of one plant proximal to the mesh-walled core into which isotopes were added, and it is probable that the roots of numerous plants would have been in close proximity to the core. As such, further 15N is likely to have been

acquired by multiple plants. Furthermore, greater 15N uptake into

plant shoots may have been recorded if the shoot tissue samples had been taken longer ater 15N addition to the mesh-walled cores.

Mesh-walled exclusion cores have been used to quantify AMF-plant nutrient dynamics in a number of studies (Johnson et al., 2001; Field et al., 2012; Field et al., 2016), and are of particular utility where the establishment of truly non-mycorrhizal control plants is not feasible, as in this study. he use of a 0.45 µm nitrocellulose membrane to exclude AMF in-growth to soil compartments is a well-established methodology in the literature (Hodge et al., 2001; Leigh et al., 2009; hirkell et al., 2016; Storer

et al., 2018), although some concerns arise in relation to the

efects of such small pore sizes on solute movement, although in the case of studies investigating mycorrhizal P uptake, such efects have been determined to be insigniicant (see Zhang et

al., 2016; Svenningsen et al., 2018). Our data show increased

plant 15N uptake in plots only where N was supplied as nitrate,

to Meridian barley, and in plots which had received high rates of N fertiliser (Figure 3). Were the movement of N through these systems determined by the porosity of the membranes used in “no access” treatments, we might expect 15N enrichment in all plots

which received 15NO

3−, which is not the case. Alternative control

treatments to disentangle the efects of AMF on plant nutrition might be tested further in future studies to determine the relative merits of each method. Non-mycorrhiza-forming mutants of a

FIGURE 4 | Percentage root length colonisation, as determined by Trypan Blue staining, was not significantly different between treatments. All inspected plants were colonised by arbuscular mycorrhizal fungi (AMF), confirmed by presence of characteristic structures, arbuscules and vesicles. Mean colonisation ranged from 28.5% in Maris Otter in Low N, to 38.0% in Meridian Low-N, but no groups were significantly different. Circles represent individual data points. High-N groups are denoted by green bars, Low-N groups are denoted by yellow bars. “N.S.D.” indicates that there were no significant differences among treatment means. Data shown are means ± SEM, n = 3.

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number of cereals have been developed (Paszkowski et al., 2006;

Watts-Williams and Cavagnaro, 2015) but to date no

mycorrhizal-defective barley mutants are available against which data from hyphal exclusion experiments can be compared. Furthermore, an AMF-colonised plant is morphologically (Gutjahr et al., 2009) and physiologically (Luginbuehl and Oldroyd, 2017) distinct from one which remains uncolonised, and comparisons between AM and mycorrhiza-defective mutants may erroneously conlate these diferences and ascribe all contrasts to the lack of mycorrhizas. Combinations of experimental approaches may be employed here to improve the rigour of ield experimentation, although the logistics of such trials may prove represent a signiicant challenge.

Identifying the mechanisms responsible for diferential nitrogen transfer from fungus to plant are beyond the scope of this study, but a number of possibilities may be considered. Numerous studies have demonstrated shits in AMF community composition or structure following N fertilisation, in grassland (Egerton-Warburton and Allen, 2000; Egerton-Warburton et al., 2007; Antoninka et al., 2011; Jiang et al., 2018) and arable systems (Verbruggen et al., 2010; Avio et al., 2013; Liu et al., 2014; Williams et al., 2017). As AMF isolates are known to be functionally diferent (Avio et al., 2006; Mensah et al., 2015) any N-driven shits in AMF community have the potential to inluence the N cycling in the system (Herman et al., 2012). Future experimental testing of the AMF community composition within cereal roots, combined with isotopic tracer studies may elucidate any link between the structure and function of AMF communities in agronomic systems.

CONCLUSIONS

Our data show that AMF transfer of N to plant hosts is inluenced by agricultural management decisions, here the cultivar of barley and the rate at which inorganic N fertiliser is supplied. he extent to which symbiotic soil microbes might enhance total nutrient uptake in the ield remains to be tested; despite demonstrating a mechanism by which plants acquire N, our data cannot indicate whether non-AMF plants in the same ield conditions might show enhanced nutrition. Further experimental investigation is required for a wider perspective on the inluence of these fungi

on their crop plant hosts, and therefore their importance in agricultural systems.

DATA AVAILABILITY STATEMENT

he datasets generated for this study are included within the supplementary information of this manuscript.

AUTHOR CONTRIBUTIONS

TT, DC, and AH designed the study. TT carried out experimental work and data analysis and wrote the initial drat of the manuscript. All authors contributed to revisions of the manuscript, and read and approved the inal submitted version.

FUNDING

his work was supported by a BBSRC White Rose DTP grant: BB/J014443/1.

ACKNOWLEDGMENTS

We thank Kate Storer and Pete Berry from ADAS for permission to use their ield trial, and for discussion about the experiment. he barley trial used here was jointly funded by AHDB and CF Fertilisers. We thank Andrew Manield, on whose land the trial was sited. We thank Heather Walker for help with the IRMS. his manuscript is an adapted and revised version of a thesis chapter submitted by Tom hirkell for the degree of Doctor of

Philosophy at the University of York, UK, in 2017. Experimental data is presented in the Supplementary Material.

SUPPLEMENTARY MATERIAL

he Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2019.01312/ full#supplementary-material

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Conlict of Interest: he authors declare that the research was conducted in the absence of any commercial or inancial relationships that could be construed as a potential conlict of interest.

Copyright © 2019 hirkell, Cameron and Hodge. his is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). he use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Contact the National Cancer Helpline on 1800 200 700, visit a Daffodil Centre or download it from www.cancer.ie Nausea and vomiting: Some chemotherapy drugs may make you.. feel